Hiroki Kawamoto

In-situ TEM observation, which has been applied in material fields such as batteries and catalysts, has recently been increasingly needed in the research fields of biotechnology and soft materials. We are already investigating the application of in-liquid TEM observation methods using specimen holders and special tools dedicated to in-liquid observation, as well as ionic liquids [ 1] ( Fig. 1) [ 2]. The in-liquid observation method enables the observation of the state of the specimen as it exists in the solvent without agglomeration or deformation of the specimen due to drying. However, as the specimen exists in liquid, clear images may not be obtained due to electron scattering by the liquid around the specimen. Therefore, Electron Scattering corrector (ES-Corrector) software [ 3], which was developed to improve the image quality of atmospheric pressure SEM, was applied to TEM images observed in liquid after studying the algorithm and setting of various parameter conditions. Fig. 1. Some experimental tools for TEM observation in liquid. A: K-kit (Optical microscope image), B: K-kit (SEM image), C: Ionic liquid on the microgrid (TEM image), D: Schematic of ionic liquid holding specimens on the microgrid. Two types of in-liquid observation tools (K-kit) and ionic liquids (IL1000) were used for in-liquid observation. For in-liquid observation of emulsions (cosmetic base), the in-liquid observation tool K-kit (Bio MA-Tek) was used; K-kit is a silicon capsule with a silicon nitride membrane. The specimen encapsulation method uses capillary action to suck up the specimen dispersion liquid inside the K-kit, seal the slit with adhesive and perform TEM observation in vacuum. Dried specimens can also be observed by evaporating the liquid. Ionic liquids were used for in-liquid observation of liposomes. An ionic liquid is a salt composed of anions and cations that shows a liquid state at room temperature.

With their wider band gaps and higher breakdown voltages, SiC and GaN are used in high-voltage power semiconductor devices. Especially the former is suitable for the application operated at a high-temperature, and its operational reliability above 200 °C which has been impossible with conventional Si semiconductors has been proven. As the SiC substrate contains various crystal defects, investigation of their behavior at high temperatures is required. Therefore, analyses in the following steps were attempted. The first is to detect polishing scratches and crystal defects (basal plane dislocations) in the SiC wafer with a Mirelis VM1000 mirror electron inspection system (Hitachi High-Tech). The mirror electron microscopy technology applied for this model enabled nondestructive defect detection without direct beam irradiation for the first time [ 1]. The second is to prepare TEM lamellae by micro-sampling the defective portion using an FIB-SEM with an FIB compatible MEMS specimen heating holder ( Fig. 1) sharing the detected coordinates on the MEMS chips and by finishing thinner for observations. The third is to observe the defects at high temperature with an HF5000 200 kV TEM/STEM (Hitachi High-Tech). The purpose of this study is to establish a feasible workflow linking these three systems. Fig. 2 depicts the analysis workflow. Image A shows the mirror electron investigation result with the locations and the types of defects on the SiC epi-wafer detected with the VM1000. Image B shows the process of extracting the desired defects by micro-sampling, loading it to a MEMS chip set in an FIB compatible MEMS specimen heating holder, and thinning it to a TEM lamella. Fig. 1 The FIB compatible MEMS Specimen Heating Holder and MEMS chip to link FIB and TEM. A: Tip shape of the holder, B: Micropillars on the MEMS chip. Schematic diagrams of its operating positions, C: for TEM observation, D: for FIB: E: for reversed TEM observation. Fig. 2 The analysis workflow. A: Mirror microscope investigation to localize stacking faults, B: TEM lamella preparation from the "Micro-sampling" to the finishing with the FIB-SEM, C: TEM observation of FIB processed TEM lamella revealing location of stacking faults.

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbotryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant without a gynoecium or stamen was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of flower bud development as well as in the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. M. lychnidis-dioicae would have a function similar to stamen-promoting factor (SPF), since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. An infection experiment also revealed that a deletion on the Y chromosome of the asexual mutant eliminated genes for maturation of tapetal cells because the tapetal cells did not mature in the asexual mutant infected with M. lychnidis-dioicae.

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia , respectively. Microbtryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae . To assess this hypothesis, an asexual mutant, without gynoecium or stamen, was infected with M. lychnidis-dioicae . A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of the flower bud development as well as the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae , but that of gynoecium development was not released. It is thought, therefore, that the suppression of gynoecium development was not released by the infection of M. lychnidis-dioicae. M. lychnidis-dioicae would have a function similar to SPF since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. Such an infection experiment also that the Y chromosome of the asexual mutant has genes related to the differentiation of archesporial cells, but none related to maturation of the tapetal cells.

When Microbotryum lychnidis-dioicae infects a male Silene latifolia, M. lychnidis-dioicae smut spores develop in the pollen sac instead of pollen. In contrast, when M. lychnidis-dioicae infects a female S. latifolia, the female flowers become male-like, promoting stamen formation. However, it is unclear when and how M. lychnidis-dioicae invades the anther. It is important to investigate not only whether hyphae exist when the apical meristem tissue differentiates into flowers and anthers, but also whether hyphae exist when stamen filaments form. We used Grocott’s methenamine silver stain and lectin stain, which stain chitin in the fungal cell wall, to search for M. lychnidis-dioicae in flower tissues. A few M. lychnidis-dioicae hyphae were observed intercellularly in the center of the connective of vascular bundles at the early anther developmental stage. Subsequently, large numbers of deeply stained M. lychnidis-dioicae hyphae were observed intercellularly in the cells surrounding the pollen sac, as well as in the center of the pollen sac. Hyphae stained with lectin were observed intercellularly in all of the stamen filaments at flower development stages. Hyphae were observed in the peduncle connecting the flower and stem. It is thought that M. lychnidis-dioicae invaded the anther via the stamen filament over a long period. Additionally, in total, 163 sections of connective were obtained, and the cell structure of each anther was colored and subjected to three-dimensional reconstruction. The M. lychnidis-dioicae hyphae observed in the connective were mainly old hyphae with large vacuoles or dead hyphae (S1 Fig). These hyphae branched out, towards the pollen sac, while growing between the cells. We also observed that the host cells that collapsed near the hyphae had thick cell walls and teliospores. Cell wall collapse and cell degeneration were observed only around hyphae with thick cell walls.

Mechanisms of suppression of pistil primordia in male flowers and of stamen primordia in female flowers differ in diclinous plants. In this study, we investigated how cell death and cell cycle arrest are related to flower organ formation in Silene latifolia. Using in situ hybridization and a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, we detected both cell cycle arrest and cell death in suppressed stamens of female flowers and suppressed pistils of male flowers in S. latifolia. In female flowers infected with Microbotryum lychnidis-dioicae, developmental suppression of stamens is released, and cell cycle arrest and cell death do not occur. Smut spores are formed in S. latifolia anthers infected with M. lychnidis-dioicae, followed by cell death in the endothelium, middle layer, tapetal cells and pollen mother cells. Cell death is difficult to detect using a fluorescein isothiocyanate-labeled TUNEL assay due to strong autofluorescence in the anther. We therefore combined a TUNEL assay in an infrared region with transmission electron microscopy to detect cell death in anthers. We show that following infection by M. lychnidis-dioicae, a TUNEL signal was not detected in the endothelium, middle layer or pollen mother cells, and cell death with outflow of cell contents, including the nucleoplast, was observed in tapetal cells.

Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving “the travelling salesman problem” and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.